TMEM139 Antibody

What is TMEM139 and what is its significance in cancer research?

TMEM139 (Transmembrane Protein 139) is a novel transmembrane protein that has been identified as significantly downregulated in non-small-cell lung cancer (NSCLC). Recent studies have shown that reduced expression of TMEM139 correlates with poor prognosis in NSCLC patients, suggesting its potential role as a tumor suppressor . The protein is predicted to be located at the plasma membrane and in focal adhesion sites, positions that are crucial for cell-cell adhesion and communication.

TMEM139's significance in cancer research lies in its ability to interact directly with E-cadherin, preventing its lysosomal degradation and subsequently inhibiting epithelial-mesenchymal transition (EMT), migration, and invasion of NSCLC cells both in vitro and in vivo . These findings suggest that TMEM139 may serve as a potential prognostic marker and therapeutic target in NSCLC and possibly other cancers.

How is TMEM139 expression regulated in non-small-cell lung cancer?

TMEM139 expression is significantly downregulated in NSCLC tumor samples compared to adjacent normal lung tissues at both mRNA and protein levels. Immunohistochemical analysis has confirmed that protein levels of TMEM139 are markedly reduced in the two main histological subtypes of NSCLC tumors (adenocarcinoma and squamous cell carcinoma) .

What are the optimal experimental conditions for using TMEM139 antibodies?

TMEM139 antibodies can be employed in multiple experimental applications with the following optimized conditions:

Western Blotting (WB):

• Recommended dilution: 1/500 - 1/2000

• Expected molecular weight: 24-30 kDa observed MW

• Buffer conditions: PBS, pH 7.3, with 0.02% sodium azide and 50% glycerol

Immunohistochemistry (IHC):

• Recommended dilution: 1/20 - 1/200

• For clinical samples: antibody dilution at 1:100 has been successfully used in paraffin-embedded NSCLC tissues

• Assessment method: Immuno-Reactive-Score (IRS) system based on staining intensity (0-3) and positive cells proportion score (0-4)

Co-Immunoprecipitation (Co-IP):

• Successfully used for both overexpressed and endogenous protein interaction studies

• For endogenous IP: anti-TMEM139 antibody at 1:200 dilution

In all applications, researchers should perform optimization experiments to determine the ideal conditions for their specific experimental setup and cell/tissue types.

How can TMEM139 antibodies be used to study EMT in cancer progression?

TMEM139 antibodies can be instrumental in studying the epithelial-mesenchymal transition (EMT) in cancer progression through multiple methodological approaches:

Protein Expression Analysis:

• Western blotting to monitor changes in EMT markers when TMEM139 is overexpressed or knocked down. Key markers include:

  • Epithelial markers: E-cadherin (increased with TMEM139 overexpression)

  • Mesenchymal markers: vimentin (decreased with TMEM139 overexpression)

  • Additional markers: type I collagen, MMP2, and MMP9 (all decreased with TMEM139 overexpression)

In vitro EMT Models:

• TGF-β1-induced EMT models can be developed using A549 cells

• TMEM139 antibodies can be used to track protein expression changes during EMT induction

• Researchers can monitor how TMEM139 overexpression inhibits the TGF-β1-induced EMT phenotype

Migration and Invasion Assays:

• Transwell migratory and Matrigel invasion assays can be performed on cells with modified TMEM139 expression

• TMEM139 antibodies can confirm expression levels in experimental and control groups

• These assays can quantitatively demonstrate that TMEM139 overexpression inhibits cancer cell migration and invasion in vitro

E-cadherin Degradation Studies:

• Cycloheximide (CHX) chase assays can be performed to study protein stability

• TMEM139 antibodies can be used to monitor both TMEM139 and E-cadherin expression over time

• This approach can verify that TMEM139 overexpression prevents E-cadherin degradation

What methodological considerations are important when studying TMEM139's interaction with E-cadherin?

When investigating the interaction between TMEM139 and E-cadherin, researchers should consider the following methodological approaches and considerations:

Co-localization Studies:

• Immunofluorescence microscopy should be employed to visualize the subcellular localization of both proteins

• Both TMEM139 and E-cadherin are located at the plasma membrane and are enriched in focal adhesion sites of A549 cells

• High-resolution confocal microscopy is recommended for precise co-localization analysis

Co-immunoprecipitation (Co-IP):

• For overexpression studies:

  • Transfect HEK-293T cells with Flag-TMEM139 and GFP-E-cadherin

  • Immunoprecipitate with anti-Flag beads followed by immunoblot analysis

• For endogenous interaction studies:

  • Use A549 and H1299 cell lines

  • Immunoprecipitate with anti-TMEM139 antibody (1:200 dilution)

  • Perform immunoblot analysis to detect E-cadherin

Protein Degradation Kinetics:

• Employ cycloheximide (CHX) chase assays to track protein degradation rates

• Use chloroquine (CQ) to inhibit lysosomal function and MG132 to inhibit proteasome-dependent degradation

• Research has shown that CQ delays E-cadherin degradation while MG132 has no effect, confirming lysosomal degradation pathway

Controls and Validation:

• Include appropriate negative controls in Co-IP experiments (IgG or unrelated antibodies)

• Verify specificity of antibodies using TMEM139 knockout or knockdown cells

• Validate protein-protein interactions using reciprocal Co-IP (pull down with E-cadherin antibody and probe for TMEM139)

How can researchers effectively use TMEM139 antibodies in immunohistochemistry for prognostic studies?

Immunohistochemistry (IHC) using TMEM139 antibodies has proven valuable for prognostic studies in NSCLC. The following methodology ensures reliable and reproducible results:

Sample Preparation:

• Surgical tumor specimens should be properly fixed and embedded in paraffin

• Section thickness should be standardized (typically 4-5 μm)

• Include both tumor and adjacent normal tissue samples for comparative analysis

Staining Protocol:

• Use anti-human TMEM139 antibody at an optimized dilution (1:100 dilution has been successfully used with Thermo Fisher PA5-57898)

• Include positive and negative controls in each batch of staining

• Employ standard antigen retrieval techniques appropriate for the specific antibody

Scoring System:

• Utilize the Immuno-Reactive-Score (IRS) system, which combines:

  • Intensity of anti-TMEM139 immunostaining (0-3 scale)

  • Positive cells proportion score (0-4 scale)

• For survival analysis, define high and low expression thresholds:

  • High expression: IRS score ≥6

  • Low expression: IRS score <6

Analysis Approach:

• Conduct scoring in a double-blind manner by two experienced pathologists

• Take five images in independent fields of view for each sample

• Use Kaplan-Meier survival analysis to correlate TMEM139 expression with patient outcomes

Data Interpretation:
The following table represents typical findings from TMEM139 expression analysis in NSCLC patients:

The data typically reveals that TMEM139 expression significantly correlates with survival outcomes but may not strongly associate with other clinical variables like gender, age, or tumor size .

What are the optimal protocols for using TMEM139 antibodies in Western blotting?

Western blotting for TMEM139 requires careful optimization to achieve reliable results. The following protocol provides a framework for effective detection:

Sample Preparation:

• Extract total protein from cells or tissues using a suitable lysis buffer

• Include protease inhibitors to prevent protein degradation

• Determine protein concentration using Bradford or BCA assay

• Load 20-40 μg of total protein per lane for optimal detection

Gel Electrophoresis:

• Use 12-15% SDS-PAGE gels due to the relatively small size of TMEM139 (observed MW: 24-30 kDa)

• Run the gel at 100-120V for appropriate separation

Transfer:

• Use PVDF membrane for better protein retention

• Transfer at 100V for 60-90 minutes or 30V overnight at 4°C

Blocking and Antibody Incubation:

• Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Incubate with primary TMEM139 antibody at dilutions of 1/500 - 1/2000

• Incubate overnight at 4°C with gentle agitation

• Wash 3-5 times with TBST, 5 minutes each

• Incubate with appropriate HRP-conjugated secondary antibody (typically 1:5000-1:10000) for 1 hour at room temperature

Detection:

• Use enhanced chemiluminescence (ECL) substrate

• Expected band size: 24-30 kDa

• If multiple bands appear, validate specificity using TMEM139 knockdown/knockout controls

Validation Steps:

• Include positive control samples (e.g., A549 or H1299 cell lines) that express TMEM139

• Consider using TMEM139-overexpressing cells as additional positive control

• For negative controls, use TMEM139 knockdown or knockout samples

How can researchers validate the specificity of TMEM139 antibodies?

Validating antibody specificity is crucial for reliable research outcomes. For TMEM139 antibodies, consider the following validation approaches:

Genetic Manipulation Methods:

• TMEM139 overexpression:

  • Transfect cells with TMEM139 expression vectors

  • Compare antibody signal between transfected and non-transfected cells

  • Observe increased signal intensity in overexpressing cells

• TMEM139 knockdown/knockout:

  • Use siRNA, shRNA, or CRISPR-Cas9 to reduce or eliminate TMEM139 expression

  • Confirm reduction or absence of antibody signal in Western blot or IHC

Multiple Antibody Validation:

• Use antibodies from different vendors or those targeting different epitopes

• Compare staining patterns and signal intensities

• Consistent results across different antibodies increase confidence in specificity

Peptide Competition Assay:

• Pre-incubate the antibody with excess immunizing peptide

• Apply to parallel samples alongside the regular antibody

• Specific antibody signal should be significantly reduced or eliminated

Cross-Reactivity Assessment:

• Test the antibody on tissues or cells known to express related proteins

• Evaluate signal in species with varying degrees of TMEM139 homology

• The TMEM139 antibody (ABIN2785261) has demonstrated reactivity with human, cow, dog, horse, pig, and rabbit samples

Correlation of Protein and mRNA Data:

• Compare protein expression patterns (using the antibody) with mRNA expression data

• Note that TMEM139 protein levels may not always correlate with mRNA levels due to post-transcriptional regulation

What are common challenges in detecting TMEM139 protein and how can they be overcome?

Researchers may encounter several challenges when detecting TMEM139 protein. Here are common issues and their solutions:

Low Signal Intensity:

• Challenge: TMEM139 is downregulated in cancer tissues, potentially resulting in weak signals.

• Solution:

  • Increase protein loading (50-80 μg)

  • Use more sensitive detection systems (ECL Plus or femto-sensitivity substrates)

  • Optimize antibody concentration and incubation time

  • Consider signal amplification techniques

Background Issues:

• Challenge: High background can obscure specific TMEM139 signals.

• Solution:

  • Increase blocking time and washing steps

  • Try different blocking agents (BSA vs. milk)

  • Reduce antibody concentration

  • Use freshly prepared buffers

Multiple Bands:

• Challenge: Detection of multiple bands near the expected molecular weight.

• Solution:

  • Validate specificity using overexpression and knockdown controls

  • Consider the possibility of post-translational modifications

  • Use different lysis buffers to preserve protein integrity

  • Include phosphatase or deglycosylation treatments if modifications are suspected

Tissue-Specific Detection Issues:

• Challenge: Variable detection across different tissue types.

• Solution:

  • Optimize fixation protocols for each tissue type

  • Adjust antigen retrieval methods based on tissue characteristics

  • Normalize data using appropriate tissue-specific controls

Storage and Antibody Stability:

• Challenge: Antibody degradation affecting detection reliability.

• Solution:

  • Store antibodies according to manufacturer recommendations (typically at -20°C)

  • Avoid repeated freeze/thaw cycles by preparing small aliquots

  • Check antibody expiration dates (typical validity is 12 months)

  • Add sodium azide (0.02%) to prevent microbial contamination

How should researchers interpret TMEM139 expression data in the context of cancer research?

Interpreting TMEM139 expression data requires careful consideration of multiple factors:

Expression Levels vs. Clinical Outcomes:

Correlation with EMT Markers:

• TMEM139 expression positively correlates with epithelial markers (E-cadherin)

• TMEM139 expression negatively correlates with mesenchymal markers (vimentin, type I collagen, MMP2, MMP9)

• Changes in these markers can help interpret the functional significance of TMEM139 alterations

Protein vs. mRNA Expression:

• TMEM139 regulates E-cadherin at the post-transcriptional level, not affecting mRNA expression

• Discrepancies between protein and mRNA levels of TMEM139 may indicate post-transcriptional regulation

• Both protein and mRNA analyses provide complementary information

Subcellular Localization:

• TMEM139 localizes to the plasma membrane and focal adhesion sites

• Changes in subcellular distribution may indicate altered function

• Co-localization with E-cadherin provides functional context

Experimental Manipulation Effects:

• TMEM139 overexpression increases E-cadherin protein levels but not mRNA expression

• TMEM139 overexpression decreases the expression of mesenchymal markers

• These effects should be consistent across different experimental models to be considered robust

What are promising areas for future TMEM139 research in cancer biology?

Several promising research directions could expand our understanding of TMEM139's role in cancer:

Mechanism of TMEM139 Downregulation:

• Investigate epigenetic mechanisms (DNA methylation, histone modifications) regulating TMEM139 expression

• Explore transcriptional regulators and signaling pathways controlling TMEM139 expression

• Study post-transcriptional regulation including microRNA targeting

Detailed Structural Studies:

• Determine the crystal structure of TMEM139 and its complex with E-cadherin

• Identify critical binding domains and amino acid residues involved in protein-protein interactions

• The N-terminal sequence (ITPVAYFFLT LGGFFLFAYL LVRFLEWGLR SQLQSMQTES PGP) could be a starting point for epitope mapping and structural analysis

Expanded Cancer Type Analysis:

• Extend studies beyond NSCLC to other cancer types

• Compare TMEM139 expression and function across different cancers

• Correlate expression patterns with cancer-specific clinical outcomes

Therapeutic Targeting:

• Develop approaches to restore TMEM139 expression in cancer cells

• Design peptide mimetics that could replicate TMEM139's interaction with E-cadherin

• Test combination therapies targeting both TMEM139 and related pathways

Detailed Lysosomal Degradation Mechanism:

• Investigate how TMEM139 prevents lysosomal degradation of E-cadherin

• Identify additional proteins involved in this regulatory pathway

• Study the "exact mechanism underlying how TMEM139-E-cadherin prevents the lysosomal degradation of E-cadherin in lung cancer cells," which remains unclear

How might single-cell analysis techniques enhance our understanding of TMEM139 in cancer heterogeneity?

Single-cell analysis techniques offer powerful approaches to understand TMEM139's role in cancer heterogeneity:

Single-Cell RNA Sequencing (scRNA-seq):

• Map TMEM139 expression across different cell populations within tumors

• Identify correlations between TMEM139 and other genes at single-cell resolution

• Discover rare cell populations with unique TMEM139 expression patterns

Single-Cell Proteomics:

• Measure TMEM139 protein levels alongside other cancer-relevant proteins

• Analyze post-translational modifications and protein-protein interactions

• Correlate protein expression with functional cellular states

Spatial Transcriptomics:

• Visualize TMEM139 expression within the tumor microenvironment

• Analyze expression patterns relative to tumor borders, vasculature, and immune cell infiltrates

• Correlate spatial distribution with invasive or metastatic potential

CyTOF and Imaging Mass Cytometry:

• Simultaneously measure TMEM139 alongside 30+ protein markers

• Create detailed protein expression maps at subcellular resolution

• Analyze correlations between TMEM139 and EMT marker expression at single-cell level

Lineage Tracing:

• Track cells with different TMEM139 expression levels during tumor evolution

• Determine if TMEM139 expression changes precede or follow metastatic events

• Study the stability of TMEM139 expression states in different microenvironments

These advanced approaches could reveal how TMEM139 expression heterogeneity contributes to tumor progression, metastasis, and treatment response, potentially identifying new therapeutic opportunities for precision medicine approaches in cancer treatment.